Multimodality molecular imaging continues to evolve into a discipline in its own right with applications to many areas of biology and clinical medicine. Although many assays exist to image intracellular proteins, cell surface proteins, reporter gene expression, as well as other cell/molecular events, no methods are currently available to image the phosphorylation status of proteins in living subjects. Phosphorylation/dephosphorylation is a key regulatory event in almost all intracellular communication networks. In particular, phosphorylation of proteins plays a key role in the insulin receptor signal (IRS) cascade and the MYC oncogene pathways. As drugs are developed to target specific signal transduction components in cancer, it will be important to have imaging assays to study the direct effects of the drugs in addition to more downstream effects (e.g., cell proliferation). We have preliminarily developed a phosphorylation sensor that utilizes split bioluminescence reporter proteins that produce low signal prior to phosphorylation and an increased signal after phosphorylation, due to split protein complementation. The specific aims of the current research proposal are to further develop and validate a new class of imaging sensors that can report on the phosphorylation status of a protein of choice and apply them to study two specific pathways. In Aim 1, we refine a recently developed strategy to detect protein phosphorylation in cell culture by using split reporter complementation. We focus on insulin receptor substrate (IRS-1) and build general vectors to explore variables that may lead to improved sensitivity of our sensors. In Aim 2, we study the IRS-1 phosphorylation sensors developed in Aim 1 in tumor models in living mice using optical and microPET technology. In Aim 3, we develop phosphorylation sensors to detect MYC protein phosphorylation so we can better study the important role of the MYC protein and anti-cancer therapies being designed to inhibit this pathway by inhibiting ERK with a drug (PD98059) soon to enter clinical trials. Finally, in Aim 4 we study optical CCD and microPET imaging of tumor models in which PD98059 is used to decrease levels of phosphorylated MYC leading to tumor regression. We will study direct effects of therapy on phosphorylation and we also study downstream effects on the tumor cells using FDG and FLT microPET imaging. In all aims, care is taken to develop generalizable approaches that should be applicable to other studies using multimodality strategies for imaging protein phosphorylation. The significance of the proposed work is that it will help to develop and validate new technologies focused on imaging protein phosphorylation in vivo. More rapid methods to validate pre-clinical models for cancer therapeutics and to translate them into the clinic will likely result.